<<

Bioprocessing Basics

JUNE 2020

Bioreactors and Fermenters: Powerful Tools for Resolving Cultivation Bottlenecks Ulrike Rasche

Advanced Technologies Facilitate Scale-up and Technology Transfer Cynthia Challener

A Beginner’s Guide to Bioprocess Modes: Batch, Fed-Batch, and Continuous Ying Yang and Ma Sha

Sponsored by

This custom ebook is sponsored by Eppendorf and presented in partnership with BioPharm International. and Fermenters: Powerful Tools for Bioreactors Scale Up, Fermentation and Fermenters Tech Transfer Resolving Cultivation Bottlenecks

SPONSORED CONTENT application note Bioreactors and Fermenters: Powerful Tools for Resolving Cultivation Bottlenecks Ulrike Rasche

Shake flasks, culture dishes, and Introduction T-flasks are the first vessels that come Many applications are well served by the to mind when we think about cultivation cultivation of or yeast in shake systems for growing eukaryotic and flasks and cells in dishes or T-flasks. prokaryotic cells in the lab. Bioreactors Bioreactors and fermenters, however, improve productivity and save work, and fermenters are another alternative time, and lab space for scientists, who: to consider if we need larger quantities • need large quantities of cells, of cells, increased efficiency of microbes, or the products cultivation, or enhanced reproducibility. they express In this white paper, we explain the key • would like to improve the characteristics of stirred-tank bioreactors reproducibility of growth, the product and which are typically grown formation, or the product quality in them. Using specific examples, • would like to systematically we demonstrate how bioreactors compare different growth conditions and fermenters can help to resolve • would like to increase the cultivation bottlenecks. cultivation efficiency

2 JUNE 2020 | BIOPHARM INTERNATIONAL SPONSORED CONTENT Bioreactors and Fermenters: Powerful Tools for Bioreactors Scale Up, Fermentation and Fermenters Tech Transfer Resolving Cultivation Bottlenecks

What are bioreactors Stirred-tank bioreactors and fermenters? Though many types of bioreactors exist, Broadly speaking, bioreactors and we will focus on stirred-tank bioreactors. fermenters are culture systems to The name is accurately descriptive. produce cells or organisms. They Cultivation takes place in the are used in various applications, tank—often called a vessel—and the including basic research and culture is mixed by stirring (instead development, and the manufacturing of shaking, for example). Stirred-tank of , food and bioreactors come in different sizes (for food additives, chemicals, and other cultures of a few milliliters to thousands of products. A broad range of cell types liters) and are made of various materials and organisms can be cultivated in (usually glass, plastic or stainless steel). bioreactors and fermenters, including The basic components and functioning cells (like mammalian cell lines, insect of stirred-tank bioreactors are always the cells, and stem cells), same. A stirred-tank bioreactor system (like bacteria, yeasts, and fungi), as well consists of several parts (Figure 1): as plant cells and . • A vessel that is filled with the Bioreactor and fermenter are two words medium in which cells are cultivated for basically the same thing. Scientists • A head plate to close the vessel who cultivate bacteria, yeast, or fungi often use the term fermenter. The • Components within or attached term bioreactor to the vessel or the head plate to often relates to Figure 1: Stirred-tank bioreactor system consisting of bio- the cultivation process control station, vessel, and bioprocess control soft- of mammalian ware. The BioFlo 120 bioprocess control station is shown cells, but is also generically used. When we talk about bioreactors in this white paper, we usually mean systems for the cultivation of microbes or mammalian cells.

3 JUNE 2020 | BIOPHARM INTERNATIONAL SPONSORED CONTENT Bioreactors and Fermenters: Powerful Tools for Bioreactors Scale Up, Fermentation and Fermenters Tech Transfer Resolving Cultivation Bottlenecks

measure and adjust the culturing is transferred from the surrounding conditions, such as feed lines air to the culture medium. This process and sensors is more efficient in shake flasks than in • A control system comprising static cultures because shaking increases external components used to exposure of the liquid surface. In adjust the culturing conditions (e.g., bioreactors, air or pure oxygen (coming for pumps) and control software example from a compressed air cylinder) is usually introduced to the culture. Creating optimal cultivation conditions With the use of spargers, the gas/liquid Like incubators and shakers, bioreactors interface can be increased and the oxygen allow for the creation of optimal supply maximized. Oxygen is important environmental conditions for the for culture growth, and the amount of growth of cells or microbes. They differ, oxygen dissolved in the medium (dissolved however, in how these are established. oxygen concentration, DO) is continuously Culture mixing. Instead of mixing by measured with a DO sensor. shaking, in a bioreactor, the culture is To keep DO at setpoint, a DO cascade stirred with an impeller. The impeller is is often set up in and executed by the mounted to the impeller shaft, which bioprocess control software. Figure 2 in turn is connected to a motor. In a shows an example for a typical DO bioreactor, not only are bacterial, yeast, cascade. If DO drops below setpoint, first and suspension cell cultures constantly the agitation speed is gradually increased mixed, but so too are the cultures of up to 1,200 rpm to increase oxygen adherent cells attached to a growth matrix. transfer from the surrounding air. If this Tempering. To obtain the right cultivation temperature, a bioreactor does not Figure 2: Dissolved oxygen cascade need to be placed inside a shaker or incubator but can remain on the lab bench. The temperature of the culture medium is continuously monitored with a temperature sensor. To regulate it, the vessel is placed in a thermowell, wrapped with a heating blanket or has a water jacket. Cooling is possible as well. Establishing aerobic or anaerobic conditions. In a shaker or incubator,

4 JUNE 2020 | BIOPHARM INTERNATIONAL SPONSORED CONTENT Bioreactors and Fermenters: Powerful Tools for Bioreactors Scale Up, Fermentation and Fermenters Tech Transfer Resolving Cultivation Bottlenecks

is not sufficient to keep DO at setpoint, incubator atmosphere is measured and the gas flow rate is increased up to three controlled, rather than the medium pH. standard liters per minute (SLPM). As a Bioreactors also differ in that a basic final measure, the oxygen concentration solution is often added to compensate in the gas mix is increased, shifting from for acidification during culture growth. gassing with air (containing 21% oxygen) For microbial cultures in bioreactors, toward gassing with pure oxygen. This basic and acid solutions are commonly is just an example. The minimum and used for pH adjustment. This is different maximum values of agitation, gas flow from cultures in shake flasks, where the rate, and oxygen concentration can be culture pH is usually not controlled. optimized depending on the Control of parameters at setpoint. In and process needs. bioreactors, different components and Anaerobic conditions can be established by the control software play together to

gassing with N2 or other anaerobic gases. control pH, temperature, and dissolved oxygen at the desired setpoint. The pH control. To regulate the pH of parameters are constantly measured carbonate-buffered media, using pH, temperature, and DO sensors. cell cultures in flasks or dishes are The sensors transmit the information to usually placed in CO incubators. In 2 the bioprocess control software, which bioreactors, the principle is the same; regulates the addition of CO and liquid CO is introduced to the culture from a 2 2 pH agents, the activity of tempering compressed gas cylinder. In bioreactors, devices, agitation, and the gassing with the medium pH is continuously air and/or O2. measured using a pH sensor and CO2 is added as needed. This is different from A typical bioprocess run the situation in a CO incubator, in which 2 To maintain cultures, scientists usually the CO concentration in the internal 2 keep them in cultivation systems within incubators. Bioreactors are usually used Sponsor’s Content for a specific experiment or production run, which may last hours, days, or BioProcessing weeks, depending on the Introduction organism and application. A bioprocess run typically comprises the following steps (Figure 3):

5 JUNE 2020 | BIOPHARM INTERNATIONAL SPONSORED CONTENT Bioreactors and Fermenters: Powerful Tools for Bioreactors Scale Up, Fermentation and Fermenters Tech Transfer Resolving Cultivation Bottlenecks

1. Preculture: The medium in the process parameter setpoints in the bioreactor is inoculated with a bioprocess control software. preculture. Often, the preculture 3. Inoculation: Once the bioreactor is is grown in a shaker or incubator. prepared, the medium is inoculated. Sometimes, smaller bioreactors are 4. Cultivation period: During the used to grow precultures for the cultivation period, agitation, pH, inoculation of larger bioreactors. temperature, and DO are typically 2. Bioreactor preparation: The monitored and controlled in real bioreactor is prepared in parallel to time via the bioprocess control inoculum preparation. Preparations software. In addition, scientists include the sterilization of often take culture samples bioreactor, feed lines, and sensors; to analyze, for example, the medium addition to the bioreactor; and the concentration the connection of the bioreactor of metabolites. Eventually, with the bioprocess control researchers feed the culture by station; and the definition of adding nutrient solutions.

Figure 3: Series of events in a typical bioprocess run

6 JUNE 2020 | BIOPHARM INTERNATIONAL SPONSORED CONTENT Bioreactors and Fermenters: Powerful Tools for Bioreactors Scale Up, Fermentation and Fermenters Tech Transfer Resolving Cultivation Bottlenecks

“Large amounts of a 5. Culture harvest: Scientists typically end the bioprocess run and harvest recombinantly expressed the culture when it enters the may be needed, for stationary growth phase. example, for biochemical or 6. Downstream processing: The structural characterization, culture broth is further processed. for ongoing use as research 7. Bioreactor cleaning: The bioreactor tools, or for ongoing use is sterilized to inactivate culture residues and cleaned. as research tools, or for evaluating medical How bioreactors and fermenters help applications.” in resolving cultivation bottlenecks Bioreactors may save work, time, and Cultures typically pass through four lab-space of scientists who need large growth phases. quantities of cells, microbes, or of the products they express. Furthermore it • In the lag phase, at the beginning can improve reproducibility of growth, of the culture, the organisms product formation, and product quality do not multiply or multiply only and increase cultivation efficiency. slowly, probably because they need to adapt to the new Culturing large amounts culture conditions. of cells and microbes • In the exponential growth phase, Sometimes, researchers require as the name says, the culture large amounts of cells or the product grows exponentially. they produce. Large amounts of a recombinantly expressed protein may be • In the stationary phase, growth needed, for example, for biochemical or stops because of the nutrient structural characterization, for ongoing concentration, the oxygen use as research tools, or for evaluating concentration, the accumulation medical applications. Small molecules of byproducts, or other factors produced by microbes and intended for become growth-limiting. use as chemical building blocks, fuels or • The stationary growth phase is food and feed additives may be required followed by the death phase, in in small quantities in the R&D phase, but which the viable cell usually large quantities are then needed density decreases. for their commercial use. Large quantities

7 JUNE 2020 | BIOPHARM INTERNATIONAL SPONSORED CONTENT Bioreactors and Fermenters: Powerful Tools for Bioreactors Scale Up, Fermentation and Fermenters Tech Transfer Resolving Cultivation Bottlenecks

of cells are needed for -based cell “It’s critical to build evidence therapy and drug research applications. during the clinical trial that Handle-less vessels a differentiated outcome is Producing cells in a couple of T-flasks or possible, whether it’s versus preparing a few liters of a bacteria or yeast existing treatments, a tablet culture in shake flasks are feasible. But if dozens or even hundreds of flasks are form, or a less optimal form needed to produce the required amount of the same treatment.” of biomass, lab space becomes limiting and the amount of manual work explodes. higher cell densities can be achieved, Stirred-tank bioreactors are scalable, sometimes making an increase of the meaning they allow increasing the size working volume unnecessary. of the cultivation vessel instead of the number of vessels, so that the work and Achieve higher cell densities space requirements stay manageable. At some point, the growth of cells and As an example: In conventional cell microbes in flasks and dishes reaches a culture consumables, CHO cells typically stationary phase. The cell concentration reach a density of 2–4 x 105 cells per cm² does not increase further. In bioreactors, (1). This corresponds to 3.5–7 x 107 cells cultures reach a stationary phase as per T-175 flask. In bioreactors, we can well (unless you perform a continuous easily reach cell densities of up to 1 x 107 bioprocess), but much higher culture cells per mL (2, 3). This corresponds to 3 densities can be achieved. 9 x 10 cells per bioreactor with a working One example: When our application volume of 250 mL. In this example, one engineers cultivated E. coli in a complex comparably small bioreactor replaces medium in a shake flask, the culture 250 T-175 flasks. entered the stationary growth phase For microbial cultures in shake flasks, the overnight and typically had an optical

situation is similar. Typically, Erlenmeyer density at 600 nm (OD600) of around flasks with capacities up to 5 L are used. 12. When they cultivated E. coli in a In contrast, bioreactors with working bioreactor, after 12 hours the culture had volumes of hundreds and thousands of also entered the stationary phase but

liters are available. reached an OD600 of 240 (2). The availability of larger vessels is not How did they manage? Using bioreactors the only advantage. In bioreactors, allowed our engineers to lessen some of

8 JUNE 2020 | BIOPHARM INTERNATIONAL SPONSORED CONTENT Bioreactors and Fermenters: Powerful Tools for Bioreactors Scale Up, Fermentation and Fermenters Tech Transfer Resolving Cultivation Bottlenecks

“Bioreactors are valuable Figure 4: DASbox Mini Bioreactors tools to optimize cultivation System allows parallel operation of up conditions.” to 24 bioreactors

the growth-limiting factors in microbial cell cultures: They improved the supply with nutrients and oxygen, as well as the temperature control (2). Fast-growing aerobic cultures consume This is just one example. Depending on lots of oxygen. If, at a high cell density, the cell line or microbial , other the culture needs more oxygen than is parameters may be critical for culture transferred to the medium, growth is growth and/or product formation, for impaired. In bioreactors, air or pure oxygen example the medium pH, metabolite can be supplied by gassing, which is more concentrations, redox potential, and efficient than shaking or agitation alone. mechanical forces. Bioreactors Growing cultures produce heat. To are valuable tools to optimize keep the temperature at setpoint, cultivation conditions. high-density bacterial cultures often Comparing growth conditions do not need to be heated up any Parallel bioprocess systems are more, but cooled down. Bioreactors available to control more than one facilitate culture cooling, in contrast to vessel (Figure 4). They often have small conventional shakers. working volumes, which helps saving If nutrients become limiting, growth stops. resources. Parallel systems have the Cultures in bioreactors can quite easily advantage that cultivation parameters and automatically fed by adding feed can be controlled independently in each solutions using the system’s integrated bioreactor. This saves time and ensures pumps. In bioprocessing we distinguish maximum reproducibility between different process modes: In a batch runs. If, for example, you would like to process the culture grows in the initially compare protein expression at eight supplied batch of medium. In a fed-batch different temperatures, in a parallel process the culture is fed to keep the bioreactor system you can perform the concentration of nutrients constant. In a experiments in parallel. This is more continuous process the culture medium is convenient than using a conventional continuously exchanged. shaker or a single bioreactor, where

9 JUNE 2020 | BIOPHARM INTERNATIONAL SPONSORED CONTENT Bioreactors and Fermenters: Powerful Tools for Bioreactors Scale Up, Fermentation and Fermenters Tech Transfer Resolving Cultivation Bottlenecks

you would either need to perform one strongly depends on the temperature. experiment after the other or would Let’s further assume that you found need several shakers or bioreactors. the optimal temperature profile for The possibility of comparing multiple growth and expression phase to process conditions in parallel make balance growth, protein expression, bioreactors well suited to systematically and aggregation. You may employ analyze the influence of several that temperature profile in a shaker; parameters on the culture outcome however, it is prone to error. Unwanted (e.g., culture growth, product formation, and unnoticed temperature fluctuations byproduct formation). In this way, may be caused by repeated opening of the shaker by other users who add researchers can gain comprehensive or remove flasks. In a bioreactor, the process understanding, which, in turn, temperature of the culture medium allows optimization of culture conditions is continuously monitored and to achieve the best possible results. adjusted as needed. As a result, the Increasing reproducibility temperature profile is maintained more In bioreactors, process parameters reliably, leading to more reproducible like pH, temperature, and dissolved results. Furthermore, the temperature oxygen can be constantly measured sensor data is recorded. Eventually, using sensors. The sensors transmit the temperature deviations are detected, information to the bioprocess control making it possible to identify the software, which regulates the action source of error. of actuators, like pumps, tempering And the situation may be much more devices, and gassing devices, to complex. Besides the temperature, keep the parameters at setpoint. The process parameters including software also continuously saves the medium pH, DO, metabolite process values, making it possible to concentrations, mechanical forces, and analyze them later. Monitoring, control, medium composition may influence and recording of process values help increasing the reproducibility of culture “Unwanted and unnoticed growth, product formation, product temperature fluctuations characteristics, and more. may be caused by repeated Let’s take a simple example. You opening of the shaker by recombinantly express a protein. Let’s assume the protein has the other users who add or tendency to aggregate and aggregation remove flasks.”

10 JUNE 2020 | BIOPHARM INTERNATIONAL SPONSORED CONTENT Bioreactors and Fermenters: Powerful Tools for Bioreactors Scale Up, Fermentation and Fermenters Tech Transfer Resolving Cultivation Bottlenecks

culture growth, product yield, protein differ and therefore differ the optimal glycosylation patterns, byproduct bioreactor design as well as setpoints formation, and more. Process of agitation, temperature, DO, pH, and parameters influence cell viability, cell other parameters. Are you cultivating behavior, and differentiation. This is mammalian cells or microorganisms? especially important if the cell itself This is the first important question when is the product of interest, for example setting up a process, and will influence if cells are used for cell therapy some basic decisions regarding certain applications. bioreactor accessories. Dependent on In a bioprocess, temperature, pH, the cell line or strain, parameters will and DO are routinely monitored and then need to be finetuned. controlled. Advanced bioprocess Bioreactor accessories control software allows the integration Although generalized, much of the of additional sensors, for example to following applies for many microbial monitor biomass and metabolites, as strains and cells. well as the setup of tailored process control strategies. Many of the microbial strains which are routinely used in industrial bioprocess Home sweet home: applications (e.g., E. coli, C. glutamicum, A bioreactor for every organism and S.cerevisiae, P. pastoris) grow much Eukaryotic cells, like mammalian cell faster than commonly used mammalian lines, stem cells, insect cells, and plant cell lines (e.g. CHO and HEK293). For cells, and microbial organisms like example, the growth rate of E. coli is bacteria, yeasts, fungi, and algae can all in the range of 1/few hours or less; the be cultivated in bioreactors. growth rate of a CHO cell line is closer to 1/day. This has important implications for However, the optimal growth conditions the design of the bioprocess system.

Sponsor’s Content Oxygen demand The oxygen demand of Production of Human fast-growing, aerobic Induced Pluripotent microbial cultures is high. Stem Cell-Derived The bioprocess system Cortical Neurospheres needs to be capable of high gas flow rates to supply enough air and/

11 JUNE 2020 | BIOPHARM INTERNATIONAL SPONSORED CONTENT Bioreactors and Fermenters: Powerful Tools for Bioreactors Scale Up, Fermentation and Fermenters Tech Transfer Resolving Cultivation Bottlenecks

more efficiently than pitched-blade or Figure 5: Two impeller types. Rush- marine impellers, but also cause higher ton-type impellers are commonly used for microbial bioprocesses. Pitch- shear forces (Figure 5). Therefore, blade impellers are commonly used the former are commonly used for for mammalian cell cultures microbial cultures, whereas the latter are commonly utilized for mammalian cell cultures.

Sterility Because of their slower growth, the contamination risk in mammalian cell or oxygen to keep DO at setpoint. cultures is much higher than in microbial To maximize the dissolved oxygen processes. The material and connection concentration in microbial cultures air/ of feed lines are two factors to consider oxygen are usually introduced to the to ensure sterility. To minimize the culture medium through submerged contamination risks in cell culture gassing, either through a dip tube with bioprocesses, many scientists prefer an open end or a porous sparger. The feed lines made of autoclavable material pores of the gassing device determine (instead of feed lines which need to be the size and number of the gas bubbles chemically cleaned) and which can be and therefore the surface available for safely connected by welding (5). gas exchange with the medium. The oxygen demand of mammalian cell Setpoints cultures is lower. Often it is not required The above paragraph provides some to maximize the gas/medium interface general recommendations regarding through sparging, but air/oxygen supply the bioreactor accessories for microbial to the bioreactor headspace is enough. and cell culture applications. But this is An important advantage of headspace not the whole story. Suitable setpoints gassing is the avoidance of shear force- for temperature, pH, DO, agitation, and causing air bubbles which may damage strategies to control them differ between sensitive mammalian cells. organisms, cell lines, and strains, and may even be different for a single Culture mixing strain used in different applications. Besides gassing, agitation is a critical Setpoints and control strategies need parameter to keep DO at setpoint. to be optimized on a case by case Rushton-type impellers mix the culture basis. Methods described in literature

12 JUNE 2020 | BIOPHARM INTERNATIONAL SPONSORED CONTENT Bioreactors and Fermenters: Powerful Tools for Bioreactors Scale Up, Fermentation and Fermenters Tech Transfer Resolving Cultivation Bottlenecks

Figure 6: Schematic representation “They can be made of of suspension cells and adherent different materials, like cells grown in stirred-tank bioreactors A) Suspension cells in stirred-tank glass, DEAE-Dextran, bioreactor B) Adherent cells grown polystyrene, and alginate.” on growth matrix in stirred-tank bioreactor glass, DEAE-Dextran, polystyrene, and alginate. There are also coated versions, whose core material is covered with , or protein mixtures like fibronectin, collagen or Matrigel®. The core material of non-coated microcarriers is not functionalized by the manufacturer, but may, however, bind proteins once the carrier is in can serve as a starting point for further contact with serum-containing culture optimization (5, 6). medium. To start a cell culture process on microcarriers, they are typically added Cultivation of adherent cells in to the culture medium at a density stirred-tank bioreactors recommended by the manufacturer and Many mammalian cell types need to subsequently the bioreactor is inoculated attach to a growth surface to survive with a single cell suspension. To support and multiply. Expansion of adherent cell attachment to the microcarrier, cells in stirred-tank bioreactors sounds the culture is only periodically agitated counterintuitive at first, but is feasible during the first few hours after if an attachment matrix is provided. The inoculation. Once the cells have attached cells attach to the matrix, which is kept to the carriers the culture is continuously in suspension by gentle agitation. agitated to keep the carriers in suspension (Figure 6). For downstream Various matrices are in common usage. processing, the microcarriers can be Microcarriers harvested and the cells detached, for Microcarriers are spherical particles example using trypsin. which provide a growth surface for Vero cells and mesenchymal stem cells adherent cells. Microcarriers typically are only two examples of adherent cells, have a diameter of 100–300 µm. They which have been cultivated in stirred-tank can be made of different materials, like bioreactors on microcarriers (7, 8).

13 JUNE 2020 | BIOPHARM INTERNATIONAL SPONSORED CONTENT Bioreactors and Fermenters: Powerful Tools for Bioreactors Scale Up, Fermentation and Fermenters Tech Transfer Resolving Cultivation Bottlenecks

“Once the cells have induced pluripotent stem cells and cells differentiated thereof (9, 10), and certain attached to the carriers tumor cell lines (11). For expansion the culture is continuously as cell-only aggregates, bioreactor agitated to keep the carriers cultures are usually inoculated with a in suspension.” single-cell suspension. Cell expansion leads to the formation and growth of Fibra-Cel® disks aggregates. The attachment of cells to Like microcarriers, Fibra-Cel disks provide each other is influenced by the agitation a growth support for adherent cells. speed and impeller type, among other Fibra-Cel disks are made of a meshwork factors. Stem cell-derived cell spheroids, of polyester and polypropylene, which neurospheres for example, can reach a is electrostatically pretreated to support remarkable degree of maturation and cell attachment. In contrast to many are promising model systems in basic microcarrier types, Fibra-Cel provides research and drug screening applications a three-dimensional growth surface (12). The absence of a synthetic matrix with a high surface-to-volume-ratio, and may simplifies downstream processing, protects cells from damaging shear for example for cell therapy applications. forces, thus increasing the total biomass Summary that can be maintained in the bioreactor. Fibra-Cel disks have a diameter of 6 The advantages of cell cultivation in mm. They provide a growth matrix in stirred-tank bioreactors, like simplified packed-bed bioreactors, and in principle scalability and improved process control can also be used free-floating in shake are not limited to suspension cells. By flasks or disposable bags. Fibra-Cel providing a growth matrix or cultivating is predominantly used for cell culture as cell-only aggregates, adherent processes for the production of secreted cells can be expanded in stirred-tank products, like recombinant proteins and bioreactors, too. viruses. They have been used for example Conclusion for the cultivation of Vero cells (7). Stirred-tank bioreactors simplify the Cell-only aggregates cultivation of large amounts of cells Instead of growing on a matrix, cells compared to conventional culture can grow in stirred-tank bioreactors systems using shakers and incubators. as cell-only aggregates. This has been Bioprocess control software facilitates described, for example, for human the precise monitoring and control of

14 JUNE 2020 | BIOPHARM INTERNATIONAL SPONSORED CONTENT Bioreactors and Fermenters: Powerful Tools for Bioreactors Scale Up, Fermentation and Fermenters Tech Transfer Resolving Cultivation Bottlenecks

critical process parameters. Cultivating Bioreactor Systems. Eppendorf Short Protocol 32. 2018. cells and microbes in bioreactors 7. Suttle A, et al. Cell Culture Bioprocesses in therefore can save the scientist work, Perfusion. BioProcess International. 17(5)si. 2019. time, and lab-space and improve the 8. Dufey et al. Expansion of Human Bone Marrow- reproducibility and efficiency of cell Derived Mesenchymal Stem Cells in BioBLU® growth and product formation. 0.3c Single-Use Bioreactors. Eppendorf Application Note 305. 2016. References 9. Olmer R, et al. Scalable Expansion of Human Pluripotent Stem Cells in Eppendorf BioBLU® 0.3 1. Camire J, et al. Adherent Growth of CHO cells in Single-Use Bioreactors. Eppendorf Application ThermoScientific cyClone SFM4CHO-A medium Note 292. 2013. using Corning® CellBIND® Surface Culture Flasks. Application Note 1.0. 2007. 10. Jiang Y, et al. Controlled, Large-Scale Manufacturing of hiPSC-Derived Cardiomyocytes 2. Suttle A and Sha M. CHO Cell Culture in in Stirred-Tank Bioreactors. Eppendorf Application Eppendorf BioBLU® 10c Single-Use Vessels. Note 409. 2019. Eppendorf Application Note 361. 2018. 11. Roldão A, et al. Culture of 3D Cell Aggregates in 3. Dorceus M. Cell Culture Scale-Up in Stirred-Tank Perfusion in a DASbox® Mini Bioreactor System. Single-Use Bioreactors. BioProcess International. Eppendorf Application Note 360. 2018. 16(11–12)si. 2018. 12. Koenig L, et al. Production of Human Induced 4. Yang Y and Sha M. A Beginner’s Guide to Pluripotent Stem Cell-Derived Cortical Bioprocess Modes – Batch, Fed-Batch, and Neurospheres in the DASbox® Mini Bioreactor Continuous Fermentation. Eppendorf Application System Eppendorf Application Note 364. 2018. Note 408. 2019. 5. Niehus A, et al. CHO Cell Cultivation in a Ulrike Rasche DASbox® Mini Bioreactor System and DASGIP® Parallel Bioreactor Systems. Eppendorf Short Eppendorf AG, Bioprocess Center, Protocol 33. 2018. Juelich, Germany 6. Niehus A, et al. E. coli Cultivation in a DASbox® Contact: [email protected] Mini Bioreactor System and DASGIP® Parallel

15 JUNE 2020 | BIOPHARM INTERNATIONAL SPONSORED CONTENT Advanced Technologies Facilitate Scale-up and Bioreactors Scale Up, Fermentation and Fermenters Tech Transfer Technology Transfer

Advanced Technologies Facilitate Scale- up and Technology Transfer Cynthia A. Challener

Single-use and modular technologies more rapidly as industries mature. plus continuous manufacturing are While the industry is increasingly important to biopharma young compared to the small-molecule scale-up and tech transfer. pharmaceutical sector, it has been of significance for several decades. Some t is hard to believe, but the of the processes that are running today biopharmaceutical industry is utilize the technologies developed when already old enough to have aging the industry was first established. I facilities that are decades old. FDA’s “Many of these processes were focus on the need for updates is creating licensed long ago and are quite complex, both opportunities and challenges for contain open steps, and are inefficient biologics manufacturers involved in in many places,” notes Parrish Galliher, the scale-up and transfer of production CTO of BioProcess Upstream at the Life technologies. Single-use and modular Sciences business of GE Healthcare. technologies, along with continuous processing approaches, are helping the FDA has recognized the need to upgrade industry both modernize old processes these older processes and facilities and is and facilities and minimize the risks imposing updates (1). Equipment suppliers associated with making significant are working closely with biopharmaceutical changes to existing systems. manufacturers to develop plans for implementing the needed changes. “This Aging facilities attract FDA attention situation provides a great opportunity Just as time passes more quickly as to update those systems, facilities, people age, it seems time goes by and practices, but also presents the

16 JUNE 2020 | BIOPHARM INTERNATIONAL Advanced Technologies Facilitate Scale-up and Bioreactors Scale Up, Fermentation and Fermenters Tech Transfer Technology Transfer

challenges associated with changing any manufacturers routinely assess single- process, including the risk of affecting use technologies as options when product quality in some way. Fortunately, new equipment is introduced to by working closely with tools and meet a process need, according to technologies suppliers and the regulators, Paul Bird, head of the manufacturing biopharmaceutical manufacturers are engineering group at Fujifilm Diosynth better positioned to overcome such ’ Billingham, UK site. challenges,” Galliher asserts. Fujifilm develops and manufactures biologics using both microbial and mammalian production systems, and “These processes have high although single-use technology is a growth rates and densities, relatively new introduction, it has had and thus the demand for an impact on mammalian manufacturing oxygen is high, making at the company. Mammalian cultures do not tend to be intensive; they do not it difficult to replicate a have high oxygen demand and do not high-productivity process grow in very high cell densities, Bird in a disposable reactor, explains. In addition, the culture growth takes place over a long period of time. according to Bird.” As a result, a reactor for mammalian cell culture is not required to have high heat Single-use solutions removal or provision for high oxygen supply; as such, single-use systems are One of the most efficacious ways to well suited. revamp older processes as required The situation is different for microbial by FDA is to upgrade with single-use cultures based on and technologies, according to Helene yeast, for example. These processes Pora, vice-president of single-use have high growth rates and densities, technologies at Pall Life Sciences. and thus the demand for oxygen is high, “Single-use systems have proven to making it difficult to replicate a high- not only reduce capital investment, productivity process in a disposable but also minimize turndown time, reactor, according to Bird. “Because resulting in more effective and higher- these cultures require very good heat quality manufacturing processes for removal, high oxygen transfer, and other scale-up and tech transfer,” she states. rigorous conditions, stainless steel tends In fact, biopharmaceutical contract to remain the best possible option for

17 JUNE 2020 | BIOPHARM INTERNATIONAL Advanced Technologies Facilitate Scale-up and Bioreactors Scale Up, Fermentation and Fermenters Tech Transfer Technology Transfer

microbial processes today,” he notes. “In addition, because the Advances in single-use technologies designed specifically for microbial running costs are less for systems may, however, lead to their single-use systems, they greater use in the future. can be considered enabling Galliher agrees that single-use technologies.” technologies are not a panacea for the upgrading of older processes. a quick and easy transition to the hybrid “Most older facilities use stainless- facility format, while for new facilities, steel manufacturing systems, so the they are making the fully flexible single- conversion to disposable technologies use facility a reality,” she says. is not automatically straightforward,” The popularity of modular systems is he observes. When single-use systems not just being driven by FDA mandates; are chosen, however, he adds that Pora also notes that manufacturers have they are fairly rapid to install and realized that modular solutions can help start-up, particularly relative to older them overcome bottlenecks and become legacy technologies, so the impact on more efficient with less investment of facilities and utilities support systems time and money. is minimized. In addition, because the running costs are less for single- The global interest in standardizing use systems, they can be considered facilities, harmonizing designs, and moving enabling technologies. to a distributed facility model with a series of smaller identical facilities around the Modular approach proves flexibility world is also driving interest in modular While modular systems are a well- systems. “For this model to be successful, established concept and have been the facilities need to be identical in order available in some form for several to facilitate tech transfer, documentation, decades, they are attracting increasing training, and essentially everything that attention in the biopharmaceutical needs to be commissioned to run these industry today. In fact, the increasing facilities in remote territories. Due to this availability of modular processing units is trend, the demand for duplicate cookie- bridging the gap for many manufacturers cutter-type modules has increased,” with both established traditional facilities Galliher comments. and new sites under construction, Another trend driving interest in modular according to Pora. “For established technologies is a reduction in production facilities, modular processing units enable volumes due to the switch to distributed

18 JUNE 2020 | BIOPHARM INTERNATIONAL Advanced Technologies Facilitate Scale-up and Bioreactors Scale Up, Fermentation and Fermenters Tech Transfer Technology Transfer

“Modular technologies “The modular nature of machinery makes modifying or adding options are also increasingly possible; their plug-and-play designs available on the production allow for easy modification with equipment level and are additional or different functions,” used to build systems Galliher observes. and processes that are Fujifilm is a good example of a biopharmaceutical manufacturer identical in different that is taking advantage of modular locations, which again production systems. In particular, the simplifies maintenance, company designed a single system for the final bulk filling of cGMP products documentation, training, that replaces numerous existing filling and validation.” systems. “By adopting a modular technology approach, we generated a production, increasing yields, and the unique capability for biologic drug product trend towards personal medicine. filling that for the first time provides “Smaller facilities require smaller flexible and adaptive manufacturing,” production systems, which makes asserts Bird. He notes that the system is modularity increasingly possible,” says flexible because the production process Galliher. He does note, however, that supports bespoke operations meeting modularity will compete with stick-built customer requirements in full, and facilities in places where labor is cheap. adaptive because the modular nature “Companies will have to consider the of the system enables point-of-use quality standards they want to reach and increases or decreases in production whether modules already constructed capacity. “This ambitious project has to meet cGMP and other industry extended the benefits for the most critical requirements will better meet their unit operation--final bulk filling--and has needs,” he adds. standardized the operator experience Modular technologies are also without impacting the flexibility to meet increasingly available on the production customer requirements,” he adds. equipment level and are used to Consistent, flexible, yet rigorous build systems and processes that business processes that introduce are identical in different locations, dependable operations on-time and which again simplifies maintenance, in full are necessary for effective documentation, training, and validation. technology transfer, according to Bird.

19 JUNE 2020 | BIOPHARM INTERNATIONAL Advanced Technologies Facilitate Scale-up and Bioreactors Scale Up, Fermentation and Fermenters Tech Transfer Technology Transfer

“In addition to effective technology The use of scale-down modeling, in transfer between R&D sites and which a large-scale system is reverse- various business units, companies engineered down to the lab scale so must have the ability to site, develop, that it can be operated to model the and deliver into manufacturing large-scale process, has also significantly correctly the first time. The reduced the difficulties associated with collaboration of highly motivated and process scale-up. With this approach, highly skilled people across multiple data that are representative of large programs of activity is the key to process behavior can be collected early success in both areas,” Bird states. in the development process and at lab-scale costs, according to Galliher. As one example of how Fujifilm is using technology to address current The use of scale-down challenges, Bird cites the company’s “ “TAG” system for digitally managing modeling, in which a large- the capture, conveyance, and retention scale system is reverse- of manufacturing knowledge to improve engineered down to the technology transfer, which provides lab scale so that it can be controlled publication and distribution of manufacturing system standards and operated to model the large- operational best practice. scale process, has also significantly reduced the Scale-down modeling for successful scale-up and tech transfer difficulties associated with One of the benefits of the use of process scale-up.” smaller production facilities is a reduction in the scale-up factor for He adds that better-designed model many biopharmaceutical processes. bioreactor, , and No longer are manufacturers required systems are allowing for even to scale-up from the lab to 20,000 smoother scale-up and tech-transfer L; more commonly processes are operations. scaled up to 2000 or sometimes 5000 Raw material choices are also being L. As a result, scale-up is less of a adjusted to improve scale-up processes. technological leap and therefore more “Instead of buying small amounts of predictable and lower-risk than in the lab quality reagents and then moving past, according to Galliher. to large-scale suppliers once a process

20 JUNE 2020 | BIOPHARM INTERNATIONAL Advanced Technologies Facilitate Scale-up and Bioreactors Scale Up, Fermentation and Fermenters Tech Transfer Technology Transfer

has been developed, today some resulted from our recent efforts,” she manufacturers are beginning the says. development process with materials Galliher sees both advantages and purchased from their eventual disadvantages associated with large-scale suppliers. This approach continuous processing, and he remains eliminates the need to change their raw uncertain whether these techniques materials at the point of tech transfer and technologies will graduate to the and scale-up and thus avoids any commercial manufacturing stage. He potential impact on performance and does, however, believe that with their product quality,” Galliher explains. knowledge and skills, service providers Continuous processing has can support biopharmaceutical manufacturers with the assessment potential to overcome scale-up of these new technologies and help and tech-transfer issues them with scale-up and tech transfer The adoption of continuous processes into their own facilities. “When you drill and intensified manufacturing may down,” concludes Pora, “the particular have a significant impact on technology challenge is reducing complexity. transfer and process scale-up. Pora believes that single-use technologies References can also be aligned with the concept 1. J. Wechsler, “Modern Manufacturing Systems Key to FDA Quality Initiative,” Pharmaceutical of continuous processing to address Technology 39 (4) 2015. scale-up issues. “Many of the recent investments at Pall have been driven by Cynthia A. Challener is a contributing editor to BioPharm International. this expectation, and our new single- pass tangential flow filtration modules This article was first published in BioPharm International, vol. 28 (6), 20-23 and systems and inline concentrator (2015). are examples of technologies that have

21 JUNE 2020 | BIOPHARM INTERNATIONAL A Beginner’s Guide to Bioprocess Modes: Batch, Bioreactors Scale Up, Fermentation and Fermenters Tech Transfer Fed-Batch and Continuous Fermentation

SPONSORED CONTENT A Beginner’s Guide to Bioprocess Modes: Batch, Fed-Batch and Continuous Fermentation Ying Yang and Ma Sha

Photo courtesy of Eppendorf Inc. In this application note, we explain the Introduction differences between batch, fed-batch, and Fermentative microorganisms continuous fermentation and how these consume carbon sources, mainly , influence culture growth. As an example, to produce various acids, alcohols, we look at E. coli fermentation processes and gases. In industry, fermentation is at bench scale. We track the biomass and used to produce biopharmaceuticals, nutrient concentrations during batch, fed- food and feed supplements, biofuels, batch, and continuous fermentation runs. and chemical building blocks. To We explain different methods to analyze establish a cost-effective process, the process, including determination bioprocess engineers have to consider of biomass, growth rate, productivity, various factors, including the costs for yield, and analysis of process costs. media and supplements, the process The comparisons can help bioprocess runtime, bacterial growth and viability, engineers to select the most appropriate product titer and yield, and product method to meet their needs. quality. The concentrations of nutrients and by-products in the culture medium In our examples we studied E. coli are important influencing factors. This fermentation at bench scale. The is why, during process development, principles may also apply to bioprocesses bioprocess engineers decide whether using other microbes or mammalian cells, to apply a batch, fed-batch or at both smaller and larger scales. continuous bioprocess.

22 JUNE 2020 | BIOPHARM INTERNATIONAL SPONSORED CONTENT A Beginner’s Guide to Bioprocess Modes: Batch, Bioreactors Scale Up, Fermentation and Fermenters Tech Transfer Fed-Batch and Continuous Fermentation

“Disadvantages are the contamination. Disadvantages are the comparatively low cell densities which comparatively low cell can be achieved and the relatively long densities which can downtime between batches, due to be achieved and the cleaning, vessel setup, and sterilization. relatively long downtime Batch fermentation is a convenient starting point for beginners in this field, between batches, due to and is often used to optimize conditions in cleaning, vessel setup, and the early stages of experimental design. sterilization.” Fed-batch fermentation is a modified version of batch fermentation. It is the In batch fermentation, microorganisms most common mode of operation in the are inoculated to a fixed volume of bioprocess industry. Microorganisms medium in a fermenter. With microbial are inoculated and grown under batch growth, the nutrients are gradually regime for a certain amount of time, then consumed and by-products accumulate. nutrients are added to the fermenter in Therefore, the culture environment increments throughout the remaining is continuously changing. The broth duration of fermentation to feed them. The is removed at the end of the run. The entire culture suspension is removed at growth curve is usually divided into the end of each run. The start of feeding distinct phases. During the initial lag is normally determined by substrate phase, growth is slow, as the organism limitation in the broth, and the time profile needs to adapt to the new environment. of feeding should be designed in a way During the exponential growth phase, that the substrate remains non-excessive the microbes divide at a constant rate. while microbial growth is fully supported. When nutrients are getting depleted Because of the addition of fresh nutrients, and by-products accumulate, growth extensive biomass accumulation normally slows down, and the culture enters the occurs in the exponential growth phase. stationary growth phase. At this point, Therefore, fed-batch fermentation is very bioprocess engineers usually harvest the useful for bioprocesses aiming for high culture. If the culture continues, it would biomass density or high product yield finally enter the death phase, which is when the desired product is positively characterized by a decrease in the viable correlated with microbial growth. cell density. Also, because the substrate is not The advantages of batch processing overfed during the process, by-product are ease of operation and low risk of accumulation is limited.

23 JUNE 2020 | BIOPHARM INTERNATIONAL SPONSORED CONTENT A Beginner’s Guide to Bioprocess Modes: Batch, Bioreactors Scale Up, Fermentation and Fermenters Tech Transfer Fed-Batch and Continuous Fermentation

“Cultures in steady-state The rate of medium exchange can be optimized to reach a steady state. In can last for days, weeks or steady state, the cellular growth rate even months, thus greatly and environmental conditions, like the reducing the downtime and concentrations of metabolites, stay making the process more constant. Cultures in steady-state can last for days, weeks or even months, economically competitive.” thus greatly reducing the downtime and making the process more In continuous fermentation, fresh economically competitive. Due to the medium is continuously added to the long cultivation, sterility maintenance fermenter, while used medium and can be challenging, and downstream cells are harvested at the same time. processing is complicated (2). Consumed nutrients are replaced and For this application note, we cultured E. toxic metabolites are removed from coli in Eppendorf BioBLU 3f Single-Use the culture. When addition and removal Vessels and directly compared these are at the same rate, the culture three modes of fermentation operation. volume stays constant. Therefore, in contrast to fed-batch fermentation, Material and methods the maximum working volume of the vessel does not limit the amount of Bacterial strain fresh medium or feed solution which The bacterial strain used in this study can be added to the culture in the was E. coli ATCC® 25922GFP™. A course of the process. Keeping the mini cell bank was prepared from working volume constant furthermore the cryovial obtained from ATCC, simplifies culture scale-up based on as described previously (2). This constant-power-to-volume strategy (1). strain has an ampicillin resistance (bla) encoded on a . A Sponsor’s Content concentration of 0.1 g/L ampicillin must be maintained in all Determination of kLa Values of Single-Use cultures, since absence or low concentrations Bioreactors of ampicillin will result in plasmid loss (3). Therefore, from

24 JUNE 2020 | BIOPHARM INTERNATIONAL SPONSORED CONTENT A Beginner’s Guide to Bioprocess Modes: Batch, Bioreactors Scale Up, Fermentation and Fermenters Tech Transfer Fed-Batch and Continuous Fermentation

“We carried out two different The chemically defined medium was prepared as follows. We prepared 10 x types of batch fermentation, citrate-phosphate buffer, 100 x trace one in complex medium element solution, thiamin (vitamin B1) and one in chemically stock solution, and magnesium sulfate definded medium. The stock solution in advance, as described in the appendix in Table A2. For each medium recipe was the only run, we first filled the vessel with a difference between these DI water solution containing citrate- two batch .” phosphate buffer and antifoam. After autoclave sterilization, we aseptically

inoculum preparation in shake flasks to added MgSO4 stock solution, 100 x fermentation runs in 3 L vessels, we trace element solution, and thiamine always added 0.1 g/L ampicillin to the stock solution to the vessel. and medium. ampicillin were also aseptically added to reach a final concentration of 90 g/L and Media preparation 0.1 g/L, respectively (Table A1). We prepared different types of media for For fed-batch fermentation, we used various purposes in this study. All chemicals chemically defined medium as well, as ® were purchased from Sigma Aldrich USA, described in Table A1. We prepared unless mentioned otherwise. 1.5 L of feeding medium containing

We carried out two different types of MgSO4, thiamine, trace elements, and batch fermentation, one in complex 614 g/L glucose. medium and one in chemically defined For continuous fermentation, the medium. The medium recipe was the preparation of chemically defined only difference between these two batch medium was almost the same as for fermentations. The complex medium fed-batch fermentation except the was a water solution containing 47.6 g/L volume for citrate-phosphate buffer and Terrific Broth Modified (Thermo Fisher the original total volume to start with Scientific®, USA), 0.4% (v/v) , (Table A1). The 1.5 L feeding medium and 0.03% (v/v) Antifoam 204 which is a was prepared the same way as for mixture of organic polyether dispersions. the fed-batch fermentation (Table A1). The detailed composition of each medium Table 1 summarizes the media prepared is described in the appendix in Table A1. for each operational mode.

25 JUNE 2020 | BIOPHARM INTERNATIONAL SPONSORED CONTENT A Beginner’s Guide to Bioprocess Modes: Batch, Bioreactors Scale Up, Fermentation and Fermenters Tech Transfer Fed-Batch and Continuous Fermentation

Table 1: Medium composition Table 2: Process parameters

overnight for inoculum growth. We transferred the entire 150 mL of growing culture from one Erlenmeyer flask to a Inoculum preparation pre-sterilized bottle, and pumped the E. coli suspension into the vessel to For inoculum preparation, 14.28 g Terrific reach an inoculation ratio of 5% (v/v) for Broth Modified and 1.2 mL glycerol each run. The starting optical density were dissolved in DI water to make a at 600 nm (OD ) was between 0.1 total volume of 300 mL, and sterilized 600 and 1.0. We had done a preliminary through autoclaving. After cooling to study to demonstrate that varying the room temperature, we transferred 150 starting biomass density between 0.1 mL of the medium and added 3 mL and 1.0 OD600 had no significant effect of the 0.2 µm membrane filtration- upon harvest in a normal 8+ hour 3 L sterilized 50 x ampicillin stock solution fermentation run of an overnight E. coli (5 g/L, Table A2) to each of the two inoculum (data not shown). 500 mL sterile Erlenmeyer flasks. We inoculated each flask with 500 µL BioBLU® 3f Single-Use Vessel E. coli suspension from a single pre- Eppendorf BioBLU 3f Single-Use thawed cryovial from the cell bank. Vessels have rigid walls that maintain We put the two Erlenmeyer flasks into the performance and scalability of an Eppendorf Innova® S44i incubator traditional stirred-tanks. BioBLU 3f shaker, set the temperature at 37 °C vessels are specifically designed for and agitation at 200 rpm, and left it microbial applications using bacteria,

26 JUNE 2020 | BIOPHARM INTERNATIONAL SPONSORED CONTENT A Beginner’s Guide to Bioprocess Modes: Batch, Bioreactors Scale Up, Fermentation and Fermenters Tech Transfer Fed-Batch and Continuous Fermentation

Figure 2: Silicone tubing connected Figure 1: Dissolved oxygen cascade with PharMed tubing

at a specific setpoint (4). In this study, we set a cascade to maintain the DO at 30%, as shown below for a DO output range between 0 and 100 (Figure 1). yeasts, and fungi. The vessels are made Since acids are produced during E. coli of an autoclavable material which allows fermentation, we only prepared 25% medium sterilization within the vessel, (v/v) ammonium hydroxide solution similar to traditional glass vessels. as the base for pH control (Table A2). In this study, we ran all fermentations For each run, 1 L base solution was using the BioBLU 3f Single-Use Vessel. prepared and sterilized through 0.2 µm The vessel has a working volume membrane filtration. We used feed lines range of 1.25–3.75 L and is equipped made of silicone for liquid addition to the bioreactor via the system’s pumps. with a macrosparger and three Rushton- For base addition, we added a section of type impellers. PharMed® tubing (Saint-Gobain®, France) Process parameters between silicone tubing connections and fitted it to the peristaltic pumps on the We used a BioFlo® 320 as the control BioFlo 320. PharMed and silicon tubing station in this study. The process were connected by straight connectors parameters applied in all fermentations and fixed with cable ties (Figure 2). are listed in Table 2. PharMed tubing better resists the base Normally in aerobic microbial than silicone tubing and by inserting it applications, DO control uses a cascade into the pump head we avoid the risk of of agitation, air flow, and oxygen flow. By damage of the mechanically stressed part setting a DO cascade, the control station of the feed line. The pump used for base automatically adjusts the assigned addition was specifically assigned as a process loops to maintain the DO level base pump through the control station.

27 JUNE 2020 | BIOPHARM INTERNATIONAL SPONSORED CONTENT A Beginner’s Guide to Bioprocess Modes: Batch, Bioreactors Scale Up, Fermentation and Fermenters Tech Transfer Fed-Batch and Continuous Fermentation

airflow cabinet. If you have bottles Figure 3: Tube welding. Silicone mounted at the base of the vessel, tubing is extended with weldable you can also autoclave them with the connectors made of C-Flex via straight connectors. A: Before welding vessel, without detaching their tubing B: After welding from the head plate. Alternatively, tubing can be connected outside a laminar air flow cabinet using a tube welder. We did all tubing connections in this study by using a SCD®-II Sterile Tubing Welder from Terumo BCT, USA as shown in Figure 3. The welding was carried out at temperatures up to 300 °C for sterility maintenance. To be welded, tubing needs to be made of weldable material like C-Flex®, or needs to be extended with a weldable connector (Figure 3).

Vessel set-up and sterilization We also prepared and sterilized 100 mL We used a pH/Redox ISM® sensor 10% (v/v) Antifoam 204 solution to (12 mm diameter with 225 mm insertion add to the ongoing fermentation when depth) for pH monitoring and an analog needed, as excessive antifoam may polarographic DO sensor (12 mm reduce the oxygen transfer rate and diameter with 220 mm insertion depth) affect microbial growth. In all the runs “We used a pH/Redox ISM® presented in this study, the 0.03% (v/v) antifoam added at the beginning of sensor (12 mm diameter medium preparation were sufficient to with 225 mm insertion control foaming throughout the entire depth) for pH monitoring culture, so no additional antifoam was and an analog polarographic added during the actual run. DO sensor (12 mm diameter Connection of addition with 220 mm insertion and harvest bottles depth) for dissolved The fermenter and addition and harvest monitoring (Mettler Toledo®, bottles can be connected under aseptic conditions, for example in a laminar Switzerland).”

28 JUNE 2020 | BIOPHARM INTERNATIONAL SPONSORED CONTENT A Beginner’s Guide to Bioprocess Modes: Batch, Bioreactors Scale Up, Fermentation and Fermenters Tech Transfer Fed-Batch and Continuous Fermentation

Figure 4: BioBLU 3F Single-Use Vessel preparation. A: Head plate of a BioBLU 3f Single-Use Vessel. Please refer to the text for details. B: BioBLU 3f Single-Use Vessel filled with a colorless medium. A pH and a DO sensor and a stainless-steel cooling finger are installed before autoclave sterilization. Feed lines are also connected before autoclaving. C: Exhaust condenser setup. The exhaust condenser should be installed post-sterilization to avoid heat damage to the head plate by the metal bracket

sample port, one thermowell, two ports “We calibrated the pH sensor for overlay liquid addition, one port for outside of the vessel, before submerged liquid addition, one gas inlet sterilization, following the with filter, one exhaust with two filters, 2-point calibration method.” one additional exhaust for pressure release during autoclaving, and four for dissolved oxygen monitoring (Mettler baffles (Figure 4). Figure 4B shows a Toledo®, Switzerland). The two sensors medium-filled BioBLU 3f vessel with a pH were installed on the head plate of sensor and a DO sensor and a stainless the BioBLU 3f vessel, through Pg 13.5 steel cooling finger, installed before ports, before sterilization of the vessel. autoclave sterilization. Figure 4C shows Figure 4 shows the setup of a BioBLU how the exhaust condenser is set up to 3f Single-Use Vessel. Its head plate has keep the two exhaust filters standing four Pg 13.5 ports which can be used for up straight. The exhaust condenser is installing DO and pH sensors, stainless- installed after autoclave sterilization to steel cooling fingers, and more. In avoid heat damage to the head plate by addition, there are one harvest tube, one the metal bracket.

29 JUNE 2020 | BIOPHARM INTERNATIONAL SPONSORED CONTENT A Beginner’s Guide to Bioprocess Modes: Batch, Bioreactors Scale Up, Fermentation and Fermenters Tech Transfer Fed-Batch and Continuous Fermentation

Sensor calibration maximum 1,200 rpm agitation until the We calibrated the pH sensor outside of DO value stabilized, to set ZERO at the vessel, before sterilization, following 0%; then we switched the gas supply the 2-point calibration method. We used from nitrogen to air at three sL/m, with 1,200 rpm agitation until the DO value a buffer at pH 7.00 to set ZERO, and a stabilized, to set SPAN at 100%. buffer at pH 4.00 to set SPAN. We calibrated the DO sensor inside the Pump calibration vessel after sterilization, just before the Pump calibration was performed before inoculation. Before vessel assembly, the run. The same tubing applied to the we filled the sensor, through its cap, peristaltic pump head for liquid addition with fresh electrolyte solution that or harvest during fermentation should was separated from the medium by a be used in pump calibration. Pump permeable membrane at the tip. Six-hour calibration was performed by pumping sensor polarization was accomplished DI water into a fully filled section of by connecting the sensor with the tubing for a set period of time and control station, which provided a voltage tracking the water volume collected in a to establish an anode and a cathode graduated cylinder at the end of tubing. within the sensor (5). The pH and Then the maximum pump speed specific temperature of the fresh medium were to the tubing used can be recorded in adjusted to 7.0 and 37 °C, respectively, the system. Pump calibration is critical, to simulate the actual E. coli culture especially for fed-batch and continuous condition. As with the pH calibration, DO fermentations in which a detailed time calibration was performed by applying profile of feeding is applied. Since the the 2-point calibration method. We BioFlo 320 has multiple bi-directional sparged pure nitrogen at three standard pumps, it is easy to simultaneously perform both feeding and harvest using liters per minute (sL/m), with the one control station, without the need for Sponsor’s Content APPLICATION NOTE No. 306 external pumps. Scale-Up of Escherichia coli Fermentation from Small Scale to Pilot Scale Using Eppendorf Fermentation Systems

Bin Li and Ma Sha Feeding strategy Eppendorf Inc., Enfield, CT, USA Contact: [email protected]

Abstract

The scale-up of fermentation processes is critical to the The parameters described include proportional vessel/ For fed-batch and success of for the production of impeller geometry, oxygen transfer rate (OTR), impeller Scale-Up of biologicals in the biopharmaceutical market. Eppendorf power numbers (Np) and impeller power consumption bioprocess systems are available with autoclavable, per volume (P/V). single-use and sterilize-in-place vessels and together We carried out E. coli fermentation runs at three cover a wide range of working volumes from less than different scales (1 L, 10 L, and 100 L) following a 1 L to as large as 2,400 L. In this application note, we constant P/V strategy, and represented the E. coli used E. coli fermentation to demonstrate the scale-up biomass growth trends by plotting optical density (at continuous fermentations, capabilities of Eppendorf fermentation systems from 600 nm, OD ) curves over time. The fermentation runs 600 Escherichia coli small scale to bench scale and pilot scale. at each of the three scales produced very similar biomass To determine suitable parameters and setpoints for yields over time, indicating excellent scalability within the operation of each fermentor, we considered critical the Eppendorf fermentor product family. scalability-related engineering parameters. Fermentation nutrient feeding and broth

Introduction harvest were carried out The ultimate goal in bioprocess development is large- scalability from geometrical perspective. The intention of this scale commercial production. Production-scale process study was to evaluate the scale-up of E. coli fermentation. To optimization is usually cost prohibitive, so optimization is do so, we first investigated engineering parameters critical often performed in smaller scale bioreactors and fermentors. for scaling up fermentation processes, such as vessel and An optimized small-scale process can then be transferred to impeller geometry, oxygen transfer rate, power number, and pilot scale following established scale-up strategies. impeller power consumption per volume. Then we utilized following a pre-set time The basics of successful fermentation scale-up start with these data to scale-up an E. coli process from small scale proportional fermentor and impeller design. Eppendorf (1 L) to pilot scale (100 L) following the constant P/V scale- fermentation systems were designed following bioprocess up strategy. industry stirred-tank design standards and provide excellent profile of pump speed. This

30 JUNE 2020 | BIOPHARM INTERNATIONAL SPONSORED CONTENT A Beginner’s Guide to Bioprocess Modes: Batch, Bioreactors Scale Up, Fermentation and Fermenters Tech Transfer Fed-Batch and Continuous Fermentation

the anticipated DO spike caused by Figure 5: Time profile of pump speed carbon source exhaustion. It is a common practice to rely on the DO spike to signal fed-batch feeding start, however, the carbon source exhaustion that causes a DO spike and may also shift E. coli metabolism and reduce peak biomass potential. For continuous fermentation, two pumps were assigned to follow the same time profile but with opposite directions, one for feeding, and the other one for harvest.

Optical density measurement can be achieved in the “Time profile” After DO sensor calibration and right setting under “Cascade” to a designated before inoculation, 20 mL of fresh medium pump (Figure 5). For time duration were taken from the vessel. One milliliter calculation, EFT (elapsed fermentation of medium was used to set blank for time) was initiated at inoculation. measurement of optical density at 600 nm The detailed time profiles of pump on an Eppendorf BioSpectrometer® speed are displayed in Tables 3 and 4 kinetic, and the rest was used to dilute the (1, 6). For highest biomass potential, we dense E. coli suspension collected in the adjusted the feeding start time before later phase during fermentation. Samples

Tables 3 & 4: Time profile of feeding pump speed at different EFT during fed- batch fermentation 3: Time profile of feeding pump at different EFT during continuous fermentation

31 JUNE 2020 | BIOPHARM INTERNATIONAL SPONSORED CONTENT A Beginner’s Guide to Bioprocess Modes: Batch, Bioreactors Scale Up, Fermentation and Fermenters Tech Transfer Fed-Batch and Continuous Fermentation

suspension to a pre-weighed 15 mL Figure 6: Batch fermentation in Eppendorf conical tube, pelleted down complex medium E. coli cells using the Centrifuge 5920 R (Eppendorf, Germany), discarded the supernatant, and weighed the pellet- containing tube again. The weight of each pellet was calculated accordingly. For cell dry weight, we transferred 5 mL of each suspension to a pre-weighed 15 mL Eppendorf Conical Tube, pelleted down using the Centrifuge 5920 R, discarded the supernatant, washed the pellet twice by resuspension and were taken every 0.5 to 1 hours until a , and finally resuspended the washed pellet to make a final decreasing trend of OD600 was observed. volume of 5 mL. We transferred 3 mL Glucose measurement of this suspension to a pre-weighed Samples taken out for optical density aluminum weighing dish, dried it at measurement were also used for 80 °C in an oven overnight for 16 hours, glucose analysis. We first pelleted the and weighed each dish again. Then we bacteria from the collected suspension calculated the dry mass of each sample. by centrifugation in a Centrifuge Results MiniSpin® plus (Eppendorf, Germany), saving the supernatants, and then For each fermentation mode, we ran the measured the glucose concentration experiments in duplicate and presented in the supernatant using a Cedex® the data obtained from both runs. Bio Analyzer (Roche Diagnostics®, Batch fermentation in complex medium Germany). is shown in Figure 6. E. coli growth

was limited, with the highest OD600 Cell wet weight (CWW) and cell dry being 11 at t = 7 h. To keep the DO at weight (CDW) measurement setpoint, only air sparging was required E. coli suspension samples with different upon reaching the maximum agitation at

OD600 ranging from 0 to 160 were taken 1,200 rpm, and no oxygen enrichment during continuous fermentation for was needed throughout the process. cell weight measurement. For cell wet We observed a standard growth curve weight, we transferred 6 mL of each for 9 h, with distinct lag, exponential

32 JUNE 2020 | BIOPHARM INTERNATIONAL SPONSORED CONTENT A Beginner’s Guide to Bioprocess Modes: Batch, Bioreactors Scale Up, Fermentation and Fermenters Tech Transfer Fed-Batch and Continuous Fermentation

Figure 7: Batch fermentation in Figure 8: High-density E. coli culture chemically-defined medium in the BioBLU 3f Single-Use Vessel. Fed batch fermentation process 11 hours post inoculation

growth, stationary, and death phase (Figure 6).

Batch fermentation in By comparing batch fermentations chemically defined medium in complex and chemically defined The growth curve of the batch medium, we found that medium fermentation in chemically defined composition plays a significant role medium is shown in Figure 7. Though in shaping the bacterial growth we observed a relatively extended and biomass potential. Since batch lag phase of 6 to 7 hours, the highest fermentation is a relatively easy

OD600 was 77 at t = 12 h, which was a operation, medium optimization can 7-fold increase of biomass compared be done in small scale batch mode to the batch fermentation in complex to reduce time and cost on process medium. The exponential growth of E. development.

coli, with a 70-fold OD600 increase, was accompanied by the drastic consumption Fed-batch fermentation of 80 g/L of glucose between 7 and In the fed-batch fermentation process

12 h. After glucose depletion at t = (Figure 8) we achieve a maximum OD600 11.5–12 h, bacterial growth stopped. For of 240 at t = 11 h. This was the highest the DO cascade, we observed oxygen cell density among all fermentations enrichment after air sparging reached in this study, which verified the 3 sL/min. Oxygen enrichment reached capability of fed-batch fermentation up to 40% toward the end of cultivation. to achieve very high biomass density.

33 JUNE 2020 | BIOPHARM INTERNATIONAL SPONSORED CONTENT A Beginner’s Guide to Bioprocess Modes: Batch, Bioreactors Scale Up, Fermentation and Fermenters Tech Transfer Fed-Batch and Continuous Fermentation

Figure 9: Fed-batch fermentation Figure 11: Continuous fermentation

throughout most of the fermentation Figure 10: Setup of the continuous process and we observed a significant fermentation process accumulation of biomass corresponding

to a more than 150-fold OD600 increase between 8 and 10 h. Glucose was depleted or almost depleted when the

maximum OD600 was reached, then it started to accumulate during death phase. For the DO cascade, oxygen demand increased to 100% at t = 10.5 h. The run during which we managed to avoid glucose depletion throughout the entire fermentation process achieved the highest peak OD.

The detailed growth curve and glucose Continuous fermentation consumption pattern are shown in The maximum OD600 observed in Figure 9. We initiated feeding before continuous fermentation (Figure 10) glucose depletion, which triggered was 159 at t = 10.5 h in one run. The strong exponential E. coli growth. With detailed growth curve and glucose the exponential increment of feeding consumption pattern are displayed rate, we avoided early glucose depletion in Figure 11. As with fed-batch

34 JUNE 2020 | BIOPHARM INTERNATIONAL SPONSORED CONTENT A Beginner’s Guide to Bioprocess Modes: Batch, Bioreactors Scale Up, Fermentation and Fermenters Tech Transfer Fed-Batch and Continuous Fermentation

Table 5: Maximum biomass Figure 12: The correlation between E. concentrations gained in different coli cell wet weight and OD 600 operation modes

t = 10.5 h. The culture volume stayed Figure 13: The correlation between E. constant throughout the culture. The OD of the harvest broth was 95. The coli cell dry weight and OD600 600 concentrations of biomass and glucose changed, meaning that the culture did not enter the steady state.

Cell wet weight, cell dry weight, and OD600 We analyzed the cell wet weight and dry weight during a continuous culture run. The corresponding cell wet weight and dry weight are measured and summarized in Figures 12 and 13. fermentation, we initiated feeding The correlations are: CWW (g/L) =

before glucose depletion, and began 2.4275 x OD600, CDW (g/L) = 0.3541

broth harvest simultaneously. Glucose x OD600.Based on the correlations, we depletion occurred at t = 7 h and calculated the maximum wet weight stayed depleted throughout the rest and dry weight of each run (Table 5). of the cultivation, indicating that the newly fed glucose was completely Specific growth rate and doubling time consumed by the growing E. coli Specific growth rate µ and cell during this period. For the DO cascade, doubling time τd can be calculated oxygen demand increased to 100% at based on biomass concentration in

35 JUNE 2020 | BIOPHARM INTERNATIONAL SPONSORED CONTENT A Beginner’s Guide to Bioprocess Modes: Batch, Bioreactors Scale Up, Fermentation and Fermenters Tech Transfer Fed-Batch and Continuous Fermentation

frame between 9 and 10 h, during which Table 6: Volumetric biomass the steepest slope in the growth curve productivity of different operation was seen. We calculated the µ to be modes max -1 0.86 h and doubling time td to be 1.16 h, meaning that during this time frame, E. coli dry mass doubled every 1.16 h. During continuous fermentation, as with fed-batch fermentation, the time frame between t = 9 h and 10 h was chosen for calculation. The maximum specific -1 growth rate µmax was 0.46 h and terms of dry weight as shown in doubling time τd was 2.17 h. equations (1) and (2) (7) Comparison of process performance We calculated the volumetric biomass productivity based on cell dry weight over the entire culture period (Table 6). Fed-batch fermentation had the highest volumetric biomass productivity at 6.27 g/(L x h) among all culture where µ is specific growth rate (h-1) modes. We also calculated the total glucose consumption and biomass between two time points t1 and t2, and X and X are biomass dry weight (g/L) yield on glucose for a more thorough 1 2 comparison among batch fermentation at time points t and t , respectively. 1 2 in chemically defined medium, fed-batch, During exponential growth, E. coli and continuous fermentation (Table 7). cells are growing at their maximum The biomass yield on glucose shows the specific growth rate, µ . The time max effectiveness of each culture method frame between t1 and t2 defines this exponential growth phase, and τd is the “During continuous biomass doubling time (h). fermentation, as with fed- We then calculated the cell doubling batch fermentation, the time for the two fastest growth rates time frame between observed in this study. During fed-batch fermentation, the maximum specific t = 9 h and 10 h was chosen growth rate occurred during the time for calculation.”

36 JUNE 2020 | BIOPHARM INTERNATIONAL SPONSORED CONTENT A Beginner’s Guide to Bioprocess Modes: Batch, Bioreactors Scale Up, Fermentation and Fermenters Tech Transfer Fed-Batch and Continuous Fermentation

Cost analysis Table 7: Biomass yield on glucose in We calculated the net cost for different operation modes growing E. coli in BioBLU 3f vessels under different fermentation modes based on the cost breakdown for chemicals, consumables, and labor from preparation to the real run and cleanup (Table 8). , equipment, and rent are not included here since they vary significantly based on the on converting glucose into bacterial actual laboratory setup. Regarding biomass, and such effectiveness is the total cost, batch fermentation is an important factor to consider, both the cheapest, especially the batch technically and economically, when fermentation in complex medium, evaluating a fermentation process. which costs 25% less than fed-batch. However, when considering the According to Table 7, the total unit cost per E. coli cell dry weight, glucose consumption in fed-batch and fed-batch is the most economic continuous fermentation was 2.6 to fermentation mode, costing 4% 3-fold higher than in batch fermentation. less than continuous fermentation, When comparing biomass yield on 64% less than batch fermentation glucose, fed-batch was the highest, in chemically defined medium, and and batch fermentation with chemically 94% less than batch fermentation in defined medium was only 11% less, complex medium. indicating its effective glucose-to- biomass conversion.

Table 8: Cost analysis of each operation mode for growing E. coli in a 3 L working volume in a BioBLU 3f Single-Use Vessel

37 JUNE 2020 | BIOPHARM INTERNATIONAL SPONSORED CONTENT A Beginner’s Guide to Bioprocess Modes: Batch, Bioreactors Scale Up, Fermentation and Fermenters Tech Transfer Fed-Batch and Continuous Fermentation

Figure 14: Growth curve comparison among four different fermentation modes

Conclusion shake flask and the chemically defined We averaged the two growth curves for medium with high glucose concentration each fermentation mode and presented in the fermenter. Fed-batch fermentation provided the highest OD at 229 on them in one graph for a clearer 600 comparison (Figure 14). average with the maximum specific growth rate at 0.86 h-1 between 9 and 10 Batch fermentation in complex medium hours and a cell doubling time of 1.16 h. lasted for only 9 hours with the lowest Significant biomass accumulation took biomass accumulation, and the maximum place early in continuous fermentation OD was 11. Batch fermentation in 600 after feeding and harvesting were chemically defined medium, fed-batch, initiated, and the specific growth rate and continuous fermentations had similar were relatively stable over 4 hours during durations, between 11 and 12.5 hours. the exponential growth with an average Batch fermentation in chemically defined maximum OD being 157. medium had the longest lag phase before 600 the exponential growth phase to reach We summarized the key findings and

a maximum OD600 at 77, which might be the advantages and disadvantages due to the significant composition change of each of the four fermentation between the complex medium in the operations in Table 9.

38 JUNE 2020 | BIOPHARM INTERNATIONAL SPONSORED CONTENT A Beginner’s Guide to Bioprocess Modes: Batch, Bioreactors Scale Up, Fermentation and Fermenters Tech Transfer Fed-Batch and Continuous Fermentation

Table 9: Comparison of batch, fed-batch, and continuous fermentation

Batch fermentation in complex glucose, indicating the robustness of the medium is a good starting point for process in converting carbon source in fermentation beginners, and it is also the medium into bacterial biomass. the recommended method in the early When high biomass and product yield stages of experimental design for are the main goal (if the product yield strain selection and culture condition is positively correlated with biomass), optimization. The growth improvement fed-batch fermentation should be observed in batch fermentation in considered, and it is also a very cost- chemically defined medium compared to effective operation. However, since the one in complex medium proves the fed-batch requires complex handling importance of medium composition in compared to batch fermentation, it fermentation. Batch fermentation using poses more challenges for an optimized medium composition is a the operator. feasible approach to achieving decent biomass without complex handling. In Continuous fermentation is an operation addition, batch fermentation in complex which is also economically competitive. medium gave a high biomass yield on Once well established with a constant

39 JUNE 2020 | BIOPHARM INTERNATIONAL SPONSORED CONTENT A Beginner’s Guide to Bioprocess Modes: Batch, Bioreactors Scale Up, Fermentation and Fermenters Tech Transfer Fed-Batch and Continuous Fermentation

Table A1: Medium preparation for different modes of fermentation in a vessel with a 3 L working volume

Table A2: Recipes for stock solutions

40 JUNE 2020 | BIOPHARM INTERNATIONAL SPONSORED CONTENT A Beginner’s Guide to Bioprocess Modes: Batch, Bioreactors Scale Up, Fermentation and Fermenters Tech Transfer Fed-Batch and Continuous Fermentation

volume, continuous fermentation can conditions, and bioprocess economy. reach a state when the specific growth Journal (9), 1503-1511. 2014. rate is purely controlled by the feeding 3. ATCC product sheet for Escherichia coli GFP (ATCC 25922GFP). Available online at rate, which is very useful for growth https://www.atcc.org/products/all/25922GFP. control in both academic research aspx#documentation. Accessed in February 2019. and industrial production. It requires 4. Mirro R. Introduction to Escherichia coli a thorough understanding of the cultivation in a stirred-tank bioreactor. bioprocess before one can successfully Eppendorf Application Note 251. 2011. carry out continuous fermentation, and 5. Willard S. A guide to calibration on the BioFlo the operator needs to deal with the 120 and BioFlo 320: dissolved oxygen sensors. Eppendorf Short Protocol 40. 2017. challenges in maintaining sterility and 6. Li B, Siddiquee K, and Sha M. The Eppendorf productivity throughout the run which BioFlo 320 bioprocess control station: an can last for weeks and months. advanced system for high-density Escherichia coli fermentation. Eppendorf Application Note Overall, depending on the experimental 340. 2015. needs and the laboratory settings, and 7. Yang Y, and Weathers P. Red light and carbon with a brief estimate of the process dioxide differently affect growth, production, budget and scheduling, we hope and quality in the microalga Ettlia oleoabundans. that this application note will help Applied and Biotechnology (99), 489-499. 2015. fermentation scientists to choose the 8. Praetorius F, Kick B, Behler KL, Honemann MN, ideal fermentation method to meet their Weuster-Botz D, and Dietz H. Biotechnological unique needs. mass production of DNA origami. Nature (552), 84-87. 2017. References 1. Li B, Sha M. Scale-up of Escherichia coli Ying Yang and Ma Sha fermentation from small scale to pilot scale using Eppendorf Inc., Enfield, CT Eppendorf fermentation systems. Eppendorf Application Note 306. 2016. USA Contact: bioprocess-info@ eppendorf.de 2. Li T, Chen X, Chen J, Wu Q, and Chen G. Open and continuous fermentation: products,

41 JUNE 2020 | BIOPHARM INTERNATIONAL SPONSORED CONTENT